My segment of this grant application is structural studies of flaviviruses and their component parts using cryo-electron microscopy (cryoEM) and X-ray crystallography. Although we will be concentrating mainly on dengue virus in the initial stages, this will lay the foundation for work on yellow fever and other flaviviruses that have a potential for being used as biological weapons. The major obstacle to structural studies of flaviviruses has been the production of a sufficient quantity of good quality virus particles and of assembly intermediates. However, these problems are being solved as we gain experience with cell culture for propagating dengue and yellow fever viruses. Our first objective is the extension of our current 26 Angstroms resolution dengue virus cryoEM map to, hopefully, about 10 Angstroms resolution. We have also already initiated a crystallographic study of the dengue virus E glycoprotein. At the same time, the membrane protein (M) and capsid protein (C) are being investigated using X-ray crystallography by Jue Chen (see Project #2) and my laboratory, respectively. Once the structures of these component proteins are known, they can then be fitted into the cryoEM maps to produce a quasi-atomic resolution, three-dimensional picture of a complete flavivirus and its assembly intermediates. In collaboration with Richard Kuhn and Tim Baker, we plan to study the immature prM particles in which the prM protein has not yet been cleaved to the pr peptide and the M membrane protein. We also intend to study the smaller, T = 1 particles that form as a by-product in the propagation of the infectious virion or as recombinant subviral particles. Other structural studies will be related to receptor (e.g. heparan sulfate) and antibody interaction with flaviviruses. In the absence of an infectious dengue virus clone, we plan to study non-infectious, virus-like particles produced by a replicon system (see Project #3 by Richard Kuhn), allowing us to study, for instance, deglycosylated particles. Locating the carbohydrate moieties will both help position the component proteins of the virus more accurately into the cryoEM density and will elucidate the function of the carbohydrate components. The replicon system will also permit the production and structural studies of mutants with defective phenotypes. It is probable that the virus will lack accurate icosahedral symmetry at acid pH required for fusion within the host cell. In collaboration with Tim Baker, we plan to develop novel methods to perform image reconstructions of cryoEM images to study fused virus-membrane complexes. The structural studies will be essential in our efforts to design inhibitors that target a variety of stages in the viral life cycle.